Alpha-haloketones are alkylating agents that are useful in a variety of contexts, including the coupling of various molecules to other, sulfhydryl-containing molecules. However, the xcex1-haloketones possess characteristics that make them problematical and impractical for use in assays and reactions in which the components of a kit for use in such methods need to be stored together and/or for a period of time. For example, the xcex1-haloketones react spontaneously with water, alkali and organic bases and therefore cannot be stored for extended periods of time in aqueous solutions, particularly in the presence of proteins at physiological pH.
xcex1-Haloketones are often not used because they are generally too reactive, cause cross-linking of the protein, and are otherwise hydrolytically unstable. Were it not for these problems, their higher reactivity would permit the use of lower concentrations of the drug derivative. Some of the xcex1-haloketones are also insufficiently soluble for use in such contexts. Thus, despite their potential usefulness in a number of diagnostic and synthetic applications, the xcex1-haloketones are often considered too unstable to be used in such applications.
Similarly, proteins are often coupled to other proteins by attaching an alkylating group to one protein and a sulfhydryl group to the other. However, it is often difficult to obtain quantitative coupling in this matter without using high concentrations of one or both protein members and/or attaching multiple or single alkylating agents to one of the proteins. Additionally, the protein having alkylating agents attached must either have no sulfhydryl group or its sulfhydryl groups must be protected to avoid polymerization. The problem again could be overcome by using a more reactive xcex1-haloketone if it were not for the difficulty of preparing and storing a protein containing a highly-reactive alkylating agent.
The present invention provides novel solutions to the foregoing problems, however, thereby rendering this class of alkylating agents practical for various diagnostic and synthetic utilities, among others. In conjunction with the disclosure of novel compounds and compositions comprising protected haloketones, the present invention also discloses methods of preparing and using protected haloketones which are useful in a variety of applicationsxe2x80x94e.g., in assays and conjugation reactions.
As an example of the latter, certain G6PDH drug conjugates are prepared by first coupling the enzyme with an xcex1-haloacid to form an xcex1-haloamide and then coupling this conjugate with a sulfhydryl-labeled drug. The method is particularly useful when the drug has a free amino group that would interfere with coupling by means of an active ester of the drug.
Therefore, in one embodiment of the invention, protecting groups that permit long-term storage of protected xcex1-haloketones, provide additional water solubility where needed, and offer a simple biocompatible method for removal are disclosed.
Therefore, in one embodiment, the invention discloses a composition comprising a protected alkylating reagent wherein deprotection of the reagent is catalyzed by an enzyme. In various preferred embodiments, the reagent includes a protecting group selected from the group consisting of a phosphate, an ester, a carbohydrate, a nucleic acid, and a lipid. Various preferred embodiments also disclose that the enzyme is selected from the group consisting of glycosidases, nucleases, lipases, esterases, hydroxylases and phosphatases.
In alternative embodiments of the invention, the reagent is a 2-halovinyl ether, a 2-halovinyl ester, a 4-halobutadienyl ether, or a 4-halobutadienyl ester. In various embodiments, the 2-halovinyl ether or ester is a 2-halovinyl monophosphate. In other embodiments, the vinyl group is substituted with one or two alkyl or aryl groups; the alkyl or aryl groups may be substituted or unsubstituted.
In various preferred embodiments of the invention, the haloketone is an xcex1-haloketone. Preferred xcex1-haloketones include xcex1-bromoacetylbenzoic acid (BABA) and xcex1-chloroacetylbenzoic acid (CABA). The compositions of the present invention may further comprise a nucleophilic agent and/or a disulfide reducing agent. Useful disulfide reducing agents include phosphines, such as tris(carboxyethyl)phosphine (TCEP).
The present invention also discloses various kits, such as kits for use in carrying out a coupling reaction. One such kit comprises, in a packaged combination, a first reagent comprising a protected alkylating reagent, in an amount sufficient to conduct at least one reaction.
In one variation, the first reagent includes a protecting group selected from the group consisting of phosphate esters, carboxylate esters, sulfate esters, glycosides and ketals. In another variation, the first reagent includes a protecting group selected from the group consisting of phosphate and carbohydrate. In various embodiments, the first reagent may be a protected haloketone and may comprise a 2-halovinyl ether or ester. An exemplary 2-halovinyl ester is a 2-halovinyl monophosphate.
A protected haloketone of the present invention preferably comprises an xcex1-haloketone, such as BABA or CABA. Alternatively, the first regent may be a 4-halobutadienyl ether or ester. In various embodiments, the vinyl group is substituted with one or two alkyl or aryl groups.
The kit may further comprise a second reagent comprising a catalyst capable of deprotecting the protected alkylating reagent. In various embodiments, the first and second reagents are included in separate containers.
In another variation of the disclosed kits, the catalyst comprises an enzyme. Suitable enzymes for use as disclosed may be selected from the group consisting of glycosidases, nucleases, lipases, esterases, hydroxylases, phosphatases (e.g., alkaline phosphatase) and ribozymes.
The present invention also discloses kits for use in a method for detecting quantitative determination homocysteine in a sample, comprising in a packaged combination: a first reagent comprising a protected alkylating reagent capable of chemically modifying homocysteine to form modified homocysteine when deprotected, a second reagent comprising an activating reagent capable of deprotecting the protected alkylating reagent, and a third reagent capable of specifically binding to the modified homocysteine, each in an amount sufficient to conduct at least one assay.
In various kits of the present invention, the first reagent comprises a protected haloketone having a phosphate protecting group. In various disclosed embodiments, the protected haloketone included in a kit is CABA or BABA. The first reagent may further comprise a homocysteine disulfide reducing agent; it may also further comprise a solid matrix coated with modified homocysteine. The modified homocysteine may be the product formed by deprotecting the protected haloketone and reaction of the deprotected haloketone with homocysteine in the sample. Alternatively, the modified homocysteine may be a 4-carboxyphenacyl thioether of homocysteine (hcy-ABA).
In various alternative embodiments, the solid matrix comprises latex beads, glass beads, a microtiter plate, nitrocellulose, agarose, liposomes, and the like. In embodiments including a homocysteine disulfide reducing agent, that agent may comprise a phosphine. One exemplary phosphine useful in the kits of the present invention is TCEP.
In other embodiments of the kits of the present invention, the second reagent further comprises a phosphatase. In one variation, the phosphatase is alkaline phosphatase. Other variations include kits wherein the second reagent further comprises a solid matrix coated with a receptor capable of specifically binding modified homocysteine. The receptor may comprise an antibody or an immunologically active fragment thereof. When the second reagent further comprises a solid matrix, that matrix may comprise latex beads, glass beads, a microtiter plate, nitrocellulose, agarose, liposomes, and the like.
In the various kits of the present invention, the matrix may further include a signaling agent affixed thereto. Various useful signaling agents include chemiluminescent agents, fluorescent agents, and chromogenic agents, to name a few examples.
In another embodiment, the invention discloses methods of preparing molecular conjugates, comprising the following steps:
(a) labeling a first molecule with a protected alkylating reagent;
(b) admixing the labeled first molecule with a second molecule, wherein the second molecule contains one or more nucleophilic groups attached thereto;
(c) adding an enzyme to the admixture of first and second molecules to initiate a coupling reaction.
In one variation, the first molecule includes an amino or hydroxyl group. The first molecule may be a small molecule or a large molecule (or polymer).
In various embodiments, the second molecule contains a sulfhydryl group. Like the first molecule, the second molecule may be a small molecule (e.g., a lipid or modified lipid) or a large molecule (or polymer). In embodiments in which the second molecule comprises a large molecule or polymer, the large molecule or polymer may be selected from the group consisting of proteins, glycoproteins, lipopolysaccharides, lipoproteins, modified saccharides, and modified nucleic acid molecules.
With respect to the protected alkylating reagent used in the methods of the present invention, that reagent is preferably a haloketone enol phosphate such as CABA (chloro acetyl benzoic acid) enol phosphate or BABA (bromo acetyl benzoic acid) enol phosphate. In another variation of the disclosed methods, the enzyme is a phosphatase, e.g., alkaline phosphatase.
The invention also discloses methods of determining the amount of homocysteine in a sample suspected of containing the homocysteine, comprising the steps of:
(a) bringing together in an aqueous medium:
(1) the sample,
(2) a first reagent comprising a protected alkylating reagent capable of being activated to chemically modify the sulfhydryl groups of homocysteine to form modified homocysteine, and
(3) a second reagent comprising a ligand capable of specifically binding to the modified homocysteine to form an immunocomplex; and
(4) a third reagent capable of activating the protected alkylating reagent.
(b) measuring the amount of the immunocomplex, the amount thereof being related to the amount of homocysteine in the sample.
In one embodiment, the first reagent further comprises a homocysteine disulfide reducing agent. In another variation, the protected alkylating reagent is a halovinyl ether or ester. One preferred embodiment provides that the halovinyl ether or ester is an xcex1-haloketone enol phosphate. Useful xcex1-haloketone enol phosphates according to the present invention include BABA enol phosphate and CABA enol phosphate.
In other disclosed variations, the third reagent is a phosphatasexe2x80x94e.g., alkaline phosphatase. Moreover, the first reagent may further comprise a solid matrix coated with hcy-ABA and/or a solid matrix coated with a receptor capable of binding modified homocysteine. In embodiments including a solid matrix, the matrix may comprise latex beads, glass beads, a microtiter plate, nitrocellulose, liposomes, agarose, and so forth. Yet another embodiment discloses that the receptor is selected from the group consisting of an antibody or an immunologically active fragment of an antibody.
In another embodiment, the invention discloses methods of determining the amount of homocysteine in a sample, wherein at least a portion of the homocysteine is in the disulfide form, comprising the steps of:
(a) preparing an admixture comprising:
(1) the sample,
(2) a releasing agent to release the homocysteine from the disulfide form,
(3) a protected alkylating reagent capable of being activated to chemically modify the sulfhydryl groups of homocysteine to form modified homocysteine, and
(4) a receptor capable of specifically binding to the modified homocysteine to form an immune complex; and
(5) an activating reagent capable of deprotecting the protected alkylating reagent.
(b) examining the medium for the amount of the immunocomplex, the amount thereof being related to the amount of homocysteine in the sample.
According to various disclosed embodiments, the releasing agent may be a phosphine (e.g., TCEP); the protected alkylating reagent may be a halogenated enol phosphate; the receptor may be an antibody or an immunologically active fragment thereof; and the activating reagent may comprise a phosphatase.
Also disclosed herein are methods of preparing a stable, protected haloketone comprising the phosphorylation of the haloketone to form its corresponding enol phosphate. In one embodiment, the protected haloketone is an xcex1-haloketone; BABA and CABA are examples of protected haloketones useful according to the present invention.
The invention further discloses a process for preparing a haloketone enol phosphate, comprising the steps of:
(a) reacting ethyl 4-acetylbenzoate and a halogenating compound to produce a halogenated acetylbenzoate ester;
(b) phosphorylating the ester in THF with phosphoryl chloride in the presence of diisopropylethylamine to produce an enol phosphoryl ester;
(c) treating the enol phosphoryl ester with aqueous sodium hydroxide to produce an enol phosphate ester sodium salt; and
(d) treating the enol phosphate ester sodium salt with sodium hydroxide to yield halovinyl enol phosphate benzoic acid sodium salt. 
In one variation, the halogenating compound is sulfurylchloride. In other variations, the halogen is selected from the group consisting of Cl, Br and I. Thus, in one embodiment, the haloketone enol phosphate is BABA enol phosphate; in another embodiment, the haloketone enol phosphate is CABA enol phosphate.
The invention also discloses that, in a method for determining the amount of homocysteine in a sample wherein the homocysteine is modified by a reagent, the improvement comprises providing a precursor to the reagent and an enzyme capable of converting the precursor to the reagent. In one variation, the precursor is a protected alkylating reagent and the enzyme is capable of deprotecting the protected alkylating reagent. In another embodiment, the reagent is an xcex1-haloketone and the precursor is an enol ester of the xcex1-haloketone. One variation discloses that the enol ester is an enol phosphate and the enzyme is a phosphatase.
The present invention also discloses a method for releasing an alkylating reagent into an aqueous medium comprising combining, in an aqueous solution,
an enol ether of an xcex1-haloketone, an enol ester of an xcex1-haloketone, an enol ether of an xcex3-halo-xcex1,xcex2-unsaturated ketone or an enol ester of an xcex3-halo-xcex1,xcex2-unsaturated ketone, and
an enzyme capable of hydrolyzing the enol ether or ester.
In one variation, the enol ester is an enol phosphate and the enzyme is a phosphatase. In another embodiment, the aqueous solution further comprises a compound that becomes alkylated subsequent to the combining step.
Further disclosed is a method of alkylating a mercaptan in an aqueous solution comprising combining the mercaptan with an enol ester of an xcex1-haloketone or the enol ester of an xcex3-halo-xcex1,xcex2-unsaturated ketone, and an enzyme capable of hydrolyzing the enol ester.
The invention also discloses a protected haloketone according to the following formulation: 
wherein R1 and R2 are alkyl, aryl or substituted alkyl or aryl; X is Cl, Br or I; and Y is a protecting group that may be removed by an enzyme.
In one embodiment, R1 is xe2x80x94C6H4COOH, R2 is H, X is Br and Y is xe2x80x94PO3H2. In another variation, R1 is xe2x80x94C6H4CONHZ, R2 is H, X is Cl and Y is xe2x80x94PO3H2. Another embodiment discloses that Z is H or NH2. Alternatively, Z is selected from the group consisting of proteins, polypeptides, oligonucleotides, polysaccharides, and lipids. In one variation of the disclosed embodiment, the enzyme is alkaline phosphatase.